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Objective To analyze the genetic characteristics of the VP1 gene of coxsackievirus A6 (CVA6) from hand, foot and mouth disease (HFMD) cases collected from Qujing prefecture in 2020. Methods (1）Virus RNA was directly abstracted from stool samples of HFMD cases and virus VP4/VP2 junction region sequence was firstly amplified and sequenced by MD91 and OL68-1 primer pairs, then the virus serotype was determined. The virus entire VP1 gene sequences were determined by relative primer pairs according to the references. ( 2) The reference sequences of CV-A6 virus entire VP1 gene were downloaded from the GenBank and the phylogenetic tree was constructed and the genetic characteristics and molecular epidemiology were analyzed. Results In 2020, a total of 49 strains of enteroviruses (EVs) were identified with a positive rate of 9.80% (49/500). Among 49 EVs strains, 48 were enterovirus species A (EV-A), accounting for 97.96% (48/49), 1 was enterovirus species B (EV-B), accounting for 2.04% (1/49). A total of 4 serotypes were identified from EV-A, among which, 3 were CV-A4, accounting for 6.12% ( 3/49); 36 were CV-A6, accounting for 73.47% ( 36/49); 5 were CV-A10, accounting for 10.20% (5/49) and 4 were CV-A16, accounting for 8.16% (4/49). Only one strain of EV-B viruses was identified (coxsackievirus B4, CV-B4), accounting for 2.04% (1/49). The phylogenetic characteristics of the entire VP1 gene sequences of 36 strains of CV-A6 identified in this study and 26 reference strains published by Yang Song and Mizuta K were analyzed by MEGA software 5.2. The results showed that all 36 Qujing strains belonged to D3a sub-genotype. Conclusion The outbreaks of HFMD in Qujing prefecture in 2020 are mainly caused by CVA6, accounting for 73.47%. Phylogenetic analysis shows that, similar to the situation in mainland China during the past years, the sub-genotype D3a of CV-A6 is the predominant virus in this outbreak. Strengthening the laboratory and molecular epidemiological surveillance is the important work in Qujing prefecture and even in the whole Yunnan province in the future. [ABSTRACT FROM AUTHOR]